Project Summary Rotator cuff tendon tears account for more than 4.5 million physician visits per year, and over 250,000 rotator cuff repair surgeries are performed annually in the United States. For massive rotator cuff defect or chronic tears with significant retraction and tissue loss, multiple strategies, including auto-, allo- and xenografts as well as synthetic implants, have been used to augment the bone-tendon junction to improve the rates of successful healing of these severe rotator cuff tears. Despite the current advances in tissue augmentation, the overall failure rate has been reported to be between 38% and 65%. Obstacles in the development of approaches to address tendon-to-bone healing are partly because (1) current augmentation options fail to mimic multizoal structure of native rotator cuff tissue; (2) uniform matrix microenvironment impedes the heterogeneous differentiation and vascularization of progenitor cells/mesenchymal stem cells (MSC); (3) limited knowledge has been gained about how MSC differentiation status and vascularization pattern within different zonal region affect rotator cuff healing. We have developed a novel strategy by combining 3D bioprinting technique with biotextile technique to generate engineered rotator cuff constructs with zonal structure and spatial bioactive factor distribution. The proposed studies will test the hypothesis that tendon-to-bone regeneration is enhanced in vitro and in vivo by spatial differentiation of adipose derived MSC (ADMSC) and spatial control of vascularization degree in pre-designed region in the optimized bioprinted microenvironment. The specific aims of the studies are (i) determine how spatial differentiation of ADMSC within bioprinted rotator cuff constructs affect tendon-to-bone healing; and (ii) determine how the spatially incorporated bioactive factors regulate ADMSC differentiation, vascularization and rotator cuff repair. A massive rabbit infraspinatus tendon defect model will be employed for both of the aims. The primary outcome measures will include inflammation, construct integration, collagen fiber alignment, collagen types in different regions, muscle quality and fat infiltration, and tensile biomechanics. This proposal will develop biological augmentation strategies to promote scarless healing. Our approach is to better understand the roles of exogenous stem cells and vasculature on tendon-to-bone interface regeneration in vitro and in vivo.